Immobilized lipase enzyme on green synthesized magnetic nanoparticles using Psidium guava leaves for dye degradation and antimicrobial activities

Zinc ferrite nanoparticles (ZnF NPs) were synthesized by a green method using Psidium guava Leaves extract and characterized via structural and optical properties. The surface of ZnF NPs was stabilized with citric acid (CA) by a direct addition method to obtain (ZnF-CA NPs), and then lipase (LP) enzyme was immobilized on ZnF-CA NPs to obtain a modified ZnF-CA-LP nanocomposite (NCs). The prepared sample’s photocatalytic activity against Methylene blue dye (MB) was determined. The antioxidant activity of ZnF-CA-LP NCs was measured using 1,1-diphenyl-2-picryl hydrazyl (DPPH) as a source of free radicals. In addition, the antibacterial and antibiofilm capabilities of these substances were investigated by testing them against gram-positive Staphylococcus aureus (S. aureus ATCC 25923) and gram-negative Escherichia coli (E. coli ATCC 25922) bacterial strains. The synthesized ZnF NPs were discovered to be situated at the core of the material, as determined by XRD, HRTEM, and SEM investigations, while the CA and lipase enzymes were coated in this core. The ZnF-CA-LP NCs crystallite size was around 35.0 nm at the (311) plane. Results obtained suggested that 0.01 g of ZnF-CA-LP NCs achieved 96.0% removal of 5.0 ppm of MB at pH 9.0. In-vitro zone of inhibition (ZOI) and minimum inhibitory concentration (MIC) results verified that ZnF-CA-LP NCs exhibited its encouraged antimicrobial activity against S. aureus and E. coli (20.0 ± 0.512, and 27.0 ± 0.651 mm ZOI, respectively) & (1.25, and 0.625 μg/ml MIC, respectively). ZnF-CA-LP NPs showed antibiofilm percentage against S. aureus (88.4%) and E. coli (96.6%). Hence, ZnF-CA-LP NCs are promising for potential applications in environmental and biomedical uses.

The utilization of green synthesis methods for nanoparticles offers several advantages compared to traditional physical and chemical procedures, based on the abundance of biological entities and the use of ecologically friendly technologies 1 .The green synthesis of nanoparticles by different macroscopic or microscopic organisms, or their derivatives, such as plant extracts, bacteria, fungi, yeast, and microalgae has recently gained an enormous interest 2 .For instance, the utilization of plants in green technology is becoming increasingly popular as an environmentally benign, non-toxic, secure, and economically advantageous alternative.Employing plant extracts to synthesize nanoparticles also provides natural components for capping agents 3 .The primary objective of green synthesis research is to enhance a synthesis technique for creating environmentally friendly, non-toxic, superparamagnetic, and cost-effective magnetic nanoparticles 5 .The synthesis and characterization of magnetic nanoparticles, which are simply defined as particles with dimensions on the nanoscale that can react to magnetic fields, are increasingly attracting attention due to their exceptional capabilities in diverse applications.Due to their magnetic properties and the fact that they are simple and safe to synthesize in a laboratory, magnetic www.nature.com/scientificreports/Psidium guava is a medicinal plant, popularly known as guava 36 .It is found across the tropical and subtropical zones and has been proven to have medicinal properties in antibacterial applications 37 , antidiarrheal 38 , antispasmodic 39 , antioxidant 40 , antiallergy 41 , antibacterial 42 , anti-cough 43 , anti-inflammatory 44 and anticancer effects 45 .Guava leaves include phytochemicals such as phenol, tannin, terpene, saponin, and flavonoids, which aid in their antibacterial capabilities 46 .Young and mature leaf extracts have been shown to inhibit both Grampositive (Staphylococcus aureus and Bacillus cereus) and Gram-negative bacteria (Salmonella enterica) 47 .
The objective of the current research is the green synthesis and characterization of zinc ferrite NPs using Psidium Guava leaves extract followed by citric acid functionalization and then immobilization of lipase enzyme to get the end nanocomposite (ZnF-CA-LP).The further objective includes investigating the potential application of the synthesized nanoparticles as photocatalysts for methylene blue degradation and also as antibacterial and antibiofilm agents for Gram-negative (E.coli), and Gram-positive (S. aureus) bacteria.

Preparation of Psidium guava leaves extract
Fresh leaves of Psidium guava were purchased from the local market in Cairo City, Egypt., followed by a thorough washing process using water to eliminate any impurities.Subsequently, the leaves were rinsed with distilled water to eliminate any contaminants.The rinsed leaves were placed within a Soxhlet apparatus and thereafter allowed to cool to ambient temperature.The solution undergoes filtration and subsequent centrifugation at a speed of 4000 (rpm) for 5 min 48 .The plant extract that has been acquired is freshly prepared for immediate utilization and can also be preserved at 4C 0 for 2-3 days, if necessary 49 .

Green synthesis of Magnetic Zinc ferrite (ZnFe 2 O 4 )
The utilization of Psidium guava leaf extract was employed in the synthesis of magnetic ZnFe 2 O 4 (ZnF) nanoparticles, serving as both a reducing agent and a capping medium.Two different solutions were prepared, consisting of 2M FeCl 3 and 1M ZnCl 2 , respectively.Subsequently, the components were combined within a flask with continuous agitation of the solution.The pH was adjusted to a value of 12 by gradually adding appropriate quantities of 1M of sodium hydroxide solution.Then a steady addition of leaf extract was implemented at a rate of 5 ml/min.The resulting mixture was subjected to continuous stirring for 15 min at a temperature of 65 °C50 .This process yielded a black precipitate composed of ZnF NPs.The particles acquired were black and demonstrated a pronounced magnetic reactivity.The material underwent a triple-washing process using ethanol and distilled water.The precipitates obtained were subjected to oven drying at a temperature of 60 °C, followed by calcination at 400 °C for 4 h 51 .These dried and calcinated precipitates were then kept for future utilization.

Citric acid-coated Zinc Ferrite magnetic nanoparticle (ZnF-CA)
Citric acid (CA) possesses the capacity to act as a stabilizer to prevent the agglomeration of particles and enhance the dispersion of nanoparticles within a solution.The ZnF NPs underwent surface modification through the direct addition of CA, resulting in the formation of modified ZnF NPs containing carboxylic groups 52 .In this experiment, ZnF NPs were combined with CA solution in water (0.02 g/ml) at a temperature of 60 °C.The mixture was stirred for 90 min.The black precipitate underwent multiple washes and was subsequently re-suspended in water with a pH level approximating neutrality, namely within the range of 7 to 7.4.The pH range used to investigate the adsorption of CA onto the magnetite surface was between 4.58 to 7.08.A solution of sodium hydroxide (1M) was employed to get a suspension pH in proximity to 7.0 53 .

Immobilization of lipase enzyme on ZnF-CA nanocomposite (ZnF-CA-LP) NCs
The immobilization process involved the utilization of a 30 mg mL -1 lipase solution in a 50 mM Tris-HCl buffer at pH 8, with the addition of 200 mg of ZnF-CA NCs.The reaction was conducted with continuous stirring at 3000 rpm for one hour at room temperature 54 .

UV-Vis spectroscopy
The UV-Vis absorption spectra of magnetic nanoparticles were measured using a UV-vis spectrophotometer (Agilent Technologies Cary 60 UV-vis) throughout the wavelength range of 200-800 nm.The samples underwent dilution and dispersion in double-distilled water under ambient conditions.The X-ray diffraction patterns were conducted using XRD-6000, Shimadzu apparatus, SSI, Japan.The radiation source employed was CuKα (40 kV, 40 mA), and a secondary beam graphite monochromator was utilized.The recorded patterns encompassed a 2-theta (2θ) range spanning from 10 to 80°, with increments of 0.02° and a counting duration of 2 s per step.

Fourier-transform infrared spectroscopy (FTIR)
The investigation of the functionalized surface of green ZnF nanoparticles and the confirmation of lipase immobilization were conducted by Fourier-Transform Infrared (FTIR) spectroscopy.This analysis was performed using an FTIR 4700 spectrometer (Jasco, Tokyo, Japan) fitted with a Peltier stabilizer DLaTGS detector.The specimens were produced using potassium bromide (KBr) pellets.The FTIR spectra were obtained within the frequency range of 400-4000 cm −1 , with a resolution of 4 cm −1 and an average of 32 scans.

Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) is a rapid and accurate method for examining the surface morphology and microstructures of a material.SEM pictures and energy-dispersive X-ray spectroscopy (EDX) data were acquired using TESCAN VEGA COMPACT SEM with Tungsten Filament as electron source and attached EDX detector JEOL JSM-6510 LV SEM Microscope (Kohoutovice, Czech Republic).The SEM was operated at a voltage of 10 kV.

Transmission electron microscopy (TEM)
The study of the shape and average size of synthesized nanoparticles was conducted using a High-resolution transmission electron microscopy (HRTEM, JEOL 3010, Japan) operated at 300 kV.The nanoparticles were evenly distributed in ethanol and applied onto a copper grid with a lacy structure using a drop-casting technique.Subsequently, the sample was allowed to dry for one hour before being subjected to examination.

Vibrating sample magnetization (VSM)
The magnetic attitude of Magnetic nanoparticles was investigated using VSM (Lakeshore 7410, USA) with an applied field from 0 Oe to 20,000 Oe at room temperature.In terms of the magnetization curve of the synthesized magnetic nanoparticles, the hysteresis loop with measurable remanence and coercivity values was observed.

Antioxidant activity of magnetic ZnF-CA-LP NPs
The antioxidant activity was conducted using D1,1-diphenyl-2-picryl hydrazyl (DPPH) technique.20 mg of ZnF-CA-LP NCs was evenly dispersed within a glass vial that already contained 1.3 mL of DPPH solution (with a concentration of 100 µmol/L in methanol) 55 .The DPPH radical exhibits a distinct violet color when dissolved in a solution, and over time, it transitions to a colorless or faint yellow shade in the presence of ZnF-CA-LP NCs.This characteristic enables convenient observation and tracking of the reaction.A negative control sample consisting of DPPH in methanol without the inclusion of the powder samples was maintained, while ascorbic acid was used as a positive control or standard for comparison testing nanoparticles.The interaction at the surface between ZnF-CA-LP NCs and DPPH reagent was facilitated by the application of gentle magnetic stirring.The resulting supernatant after centrifugation was collected at 15-min intervals for analysis using UV-VIS spectroscopy at 517 nm against the methanol as blank.Each sample was analyzed in triplicate 56 .The percentage of inhibition was calculated against blank: where A 0 is the absorbance of the DPPH solution and A 1 is the absorbance of the sample at specific time.

Photocatalytic activity measurement
In this experimental study, a total of 10 mg of several types of nanoparticles (ZnF NPs, ZnF-CA NPs, and ZnF-CA-LP NCs were solubilized in a 50 ml aqueous solution containing Methylene blue (MB) with an initial concentration of 10.0 ppm.The solubilization process was carried out under continuous stirring.Then, the mixture was placed in a light-restricted chamber for 30 min to establish a state of equilibrium between the processes of adsorption and desorption of the photocatalyst and methylene blue dye.After the completion of the solution, it is subjected to irradiation using a UV lamp.At regular intervals of 15 min, a volume of 2.0 ml of the sample was extracted from the tube and subjected to centrifugation to facilitate the separation of the photocatalyst.The absorbance of the solution was measured at a wavelength of 664.0 nm using a UV-Visible spectrophotometer 57 .The proportion of degradation was determined by the utilization of a mathematical equation.
where C i represents the initial absorption of the dye and C f represents its absorption following a different time (min).The identical operation is conducted using variables such as pH, dye concentration, catalytic dose, and presence of H 2 O 2 .

Reusability of Magnetic ZnF-CA-LP NCs
The reusability of ZnF-CA-LP NCs was assessed by conducting multiple consecutive operation cycles for photocatalytic reduction of MB dye when subjected to UV irradiation for 120 min.After every 120 min, a sample of www.nature.com/scientificreports/ the dye solution was withdrawn and analyzed for residual dye concentration by the UV-Vis spectrophotometer, and the Nanocomposite was separated with the magnet.The ZnF-CA-LP NCs were thoroughly rinsed with water.Subsequently, its reusability was used by putting it into another MB solution to initiate a fresh cycle.The ZnF-CA-LP NCs activity was quantified in each cycle and expressed as a relative value.

Antimicrobial activity and minimal inhibitory concentration (MIC)
Both the minimum inhibitory concentration (MIC) and the disk diffusion method on agar were employed in the antibacterial studies.The study involved the examination of two separate strains, specifically one strain classified as gram-positive (S. aureus ATCC25923), and another strain classified as gram-negative (E. coli ATCC25922).The broth solution was supplied with each strain at a concentration of 0.5 MacFarland for the subsequent dilution operation.The method employed to ascertain the lowest concentration required to hinder the proliferation of microorganisms was referred to as the MIC 58 .
To investigate the effects of different nanoparticle concentrations in Mueller-Hinton (MH) broth, a series of experiments were conducted in triplicate.These experiments were carried out using 96-well plates 59 .A study was done wherein three various types of inhibition controls were employed.The positive inhibition control consisted of Gentamycin at a concentration of 10 µg/ml.The negative control was established by DMSO.The microplates were subjected to an incubation period lasting 48 h, during which a consistent temperature of 37 °C was maintained.The optical density at 600 nm was measured using a primary filter and a microplate spectrophotometer 60 .
The Kirby-Bauer method was implemented during the disc diffusion examination.The microorganism inoculum was evenly distributed across the entire surface of the Mueller-Hinton agar in a petri dish 61 .Subsequently, a 10 µg/ml nanoparticle layer was evenly distributed across the surface of the agar.The containers were subjected to an incubation period of 24 h at a temperature of 37 °C.Whether or not the discs were encircled by a halo of growth inhibition was utilized to assess the outcomes.

Antibiofilm activity of the synthesized ZnF-CA-LP NCs
Biofilms, composed of polysaccharides, proteins, and nucleic acids, serve as barrier systems against pathogens and infections 62 .A qualitative evaluation of biofilm formation was performed by visually examining the biofilm that developed on the inner surface of the tube 63 :.The antibiofilm efficacy of the ZnF-CA-LP NCs of a concentration of 10 µg/ml was evaluated with a control sample, with a specific focus on susceptible bacterial strains without the presence of ZnF-CA-LP NCs and with the presence of ZnF-CA-LP NCs.A quantity of five milliliters of nutrient broth was inserted into the tubes, subsequently followed by the introduction of an aliquot containing 0.5 McFarland of the bacteria being studied 64 .The tubes were thereafter placed in an incubator set at a temperature of 37 0 C for a duration of 24 h.
The contents of the treated and untreated containers were extracted and subsequently submitted to treatment with Phosphate Buffer Saline (PBS) at a pH = 7, followed by dehydration.The bacterial layers were rendered stable for 10 min by treating them with a solution containing 3.0% sodium acetate (5 ml), Subsequently, they were rinsed using deionized water.
The biofilms were subjected to a staining procedure utilizing a 0.1% concentration of Crystal Violet (CV) for 15 min.Following that, the biofilms underwent a washing procedure using deionized water to remove any remaining discoloration.To promote the extraction of the stain, a volume of 2.0 ml of ethanol was combined.The observation of a visible pigmented coating on both the upper and lower surfaces of the tube indicated the formation of a biofilm with a favorable result.The quantification of bacterial biofilms was performed using a UV-Vis spectrophotometer, which was adjusted at a wavelength of 580.0 nm.The determination of the percentage of inhibition was carried out using the designated equation 65 .

Statistical analysis
The statistical analysis of our results was conducted utilizing the ONE-WAY ANOVA (with a significance level of (P < 0.05).Specifically, we employed Duncan's multiple range test and the least significant difference (LSD) method for summarizing the findings.The investigation and evaluation of the outcomes and data were conducted using SPSS version 15.Data were processed in the Origin Pro 8.5 SR1 software.

UV-Vis spectroscopy analysis
Figure 1a shows UV-visible spectra of an aqueous solution of the crude extract.The extract shows a broad absorption peak at the wavelength of 279 nm, which is assigned to the characteristic absorption of the phenolic groups 66 .Figure 1b displayed the distinct continuous peak absorption pattern of the synthesized magnetite nanoparticles (ZnF, ZnF-CA, ZnF-CA-LP) within the visible spectrum, specifically between 300 and 800 nm.From the absorption spectra pure zinc ferrite nanoparticles are observed in the UV-visible region of 200-500 nm 67 .Our results show that the peak of UV-vis spectrum of ZnF NPs is at 350 nm.After the lipase is immobilized on ZnF-CA NPs CA using carbodiimide activation of the citric acid carboxylic acid groups, the peak of UV-vis spectrum of ZnF-CA-LP NCs is shifted to 260 nm.Fourier transform infrared spectroscopy analysis Figure 2 illustrates the FTIR absorption spectra of the synthesized ferrite samples.The absorption peak observed at 540 cm -1 and 450 cm -1 in ZnF is due to the Fe-O and Zn-O vibration in the normal sample respectively.The presence of a broad peak at 3450 cm -1 can be attributed to the existence of structural hydroxyl (OH) groups, as well as the presence of molecular water traces.Moreover, the prominent peak observed at 1370 cm -1 can be attributed to the C-H vibration band, as well as the peaks observed at 1456 and 1624 cm -1 , which is attributed to the symmetric stretching of the C = O bond in the COOH group of citric acid, this indicates the binding of a citric acid radical to the surface of ZnF NPs.The presence of bending vibration peaks at 1153 and 1020 cm -1 in the CO=N-H amide I region indicates that lipase is immobilized on ZnF-CA (referred to as ZnF-CA-LP) 67,68 (Fig. 2).

X-ray diffraction analysis
The X-ray diffraction pattern of ZnF NPs and ZnF-CA-LP NCs, as obtained, are depicted in Fig.  where, D is the crystallite size, λ is the X-ray wavelength used, β is the full width at half maximum (FWHM) and θ is the diffraction angle.The ZnF-CA-LP NCs crystallite size was 35.0 nm at the (311) plane, which was the strongest peak.The performance of nano photocatalytic substances is greatly impacted by particle size.

Scanning electron microscopy analysis
The morphology of the magnetic nanoparticles was examined using TESCAN VEGA COMPACT SEM.

Transmission electron microscopy analysis
The synthesized Naked ZnF NPs exhibit a semi-spherical morphology and are characterized by tiny dimensions, as depicted in Fig. 5A.The measured sizes ranged from 20.5 to 45.0 nm, with an average particle size of 25.0 nm.In contrast, ZnF-CA NPs typically exhibit a spherical morphology characterized by smooth surfaces and a narrow range of sizes from 25.0 to 50.0 with an average diameter of 32.5 nm, Fig. 5B. Figure 5C shows the typical HRTEM images of ZnF-CA-LP NCs. Figure 5D denotes the [311] crystalline facet of the crystal with an interplanar distance of 0.258 nm.The HRTEM images reveal that the particles exhibit a spherical morphology and possess a consistent size distribution.The size of these particles can be adjusted within the range of 30.5-55.5 nm, with an average diameter of 38.50 nm which is larger than the synthesized nacked ZnF nanoparticles due to the loading of CA and lipase on their surface.

Magnetic measurements
The magnetic performance of the prepared nanoparticles was also studied with the help of the Vibrating sample magnetometer (VSM), The superparamagnetic properties of the nanoparticles have been confirmed through the absence of the hysteresis loop 73 , as shown in Fig. 6.The Magnetization (Ms) and Remanence (Mr) of ZnF nanoparticles (NPs) are higher compared to that of ZnF-CA NPs and ZnF-CA-LP measuring approximately (15.3, 0.11), (10.3,0.094) and (5.56,0.027)emu/g respectively.While the coercivity (Hc)values for ZnF (37.6 Oe,), ZnF-CA (88.9 Oe), and ZnF-CA-LP (111.08 Oe) are also reported, Table 2.

Antioxidant activity
It is noticed that in the presence of ZnF-CA-LP NCs the color of DPPH solution gradually changes from deep violet to pale yellow.Figure 7b displays the UV-visible spectrum of DPPH at various time intervals in the presence of ZnF-CA-LP NCs.The DPPH scavenging activity of these nanoparticles is determined by measuring the reduction in absorbance at 517 nm 74 .The intensity at 517 nm decreases progressively over time, providing proof of the ZnF-CA-LP NCs' ability to scavenge free radicals.This is illustrated in Fig. 7a.The experiment yielded a result indicating that the ZnF-CA-LP NCs exhibited a DPPH scavenging activity of 62.8% compared to ascorbic acid (positive control) activity of 84.4 within 120 min.ZnF-CA-LP NCs possess the ability to transfer their electron density to the free radical situated at the nitrogen atom in DPPH.

Screening of photocatalytic performance of prepared nanocatalysts
The photocatalytic efficacy of the synthesized samples was determined by assessing their ability to degrade an aqueous solution of Methylene Blue dye (MB, C 16 H 18 ClN 3 S) 75 under the influence of UV irradiation.The maximum absorption band of Methylene Blue (MB), which was determined at a wavelength of 664 nm 76 , exhibited a steady reduction in intensity over time and upon exposure to ultraviolet (UV) radiation, Fig. 8a. Figure 8b represents the calibration curve of MB obtained by different concentration ranged from 2.5 to 15 ppm.Upon

Effect of initial pH
This section focuses on the investigation of the impact of pH on the process of photocatalytic degradation.The pH values in this study range from 5.0 to 9.0, and the experiments were conducted at the ambient temperature (25 °C).Fifty ml of MB solution with a concentration of 10.0 ppm was subjected to stirring.Figure 9a illustrates the influence of the initial pH values on the photodegradation of MB under irradiation for 120 min.The findings indicated that the removal of MB was enhanced at higher pH values 77 .The degradation of MB is a multistage process, with the initial phase including the adsorption of MB onto the catalyst.The surface electrical properties of magnetic nanoparticles (MNPs) were found to be responsible for their photocatalytic potential, which was influenced by the presence of several interlayer anions 78 .The enhanced surface potential of ZnF-CA-LP NCs facilitated the adsorption of MB, hence enhancing the efficiency of charge generation induced by light.The initial pH level has the potential to alter both the surface charge and agglomeration of photocatalysts.

Point of zero charge
The determination of the point of zero charge (PZC) involved the addition of 10 mg of ZnF-CA-LP NCs to a 50 mL solution of 0.01 M NaCl.The pH values of the solutions were adjusted to 2, 4, 6, 8, 10, and 12 with the addition of HCl or NaOH.The samples were subjected to agitation at a speed of 200 rpm for 48 h.After the ZnF-CA-LP NCs were subjected to magnetic separation, the pH values of the solutions were determined.The pH at the PZC was determined by utilizing a graph that correlates the initial pH with the end pH 79 .According to Fig. 9b, it can be observed that the pH at the PZC was identified as 6.8, as there was no substantial disparity between the final and initial pH readings.This observation indicates that the surface charge of the photocatalyst is positive when the pH is lower than the point of zero charge (PZC), and negative when the pH is higher than the PZC.Moreover, when the pH of the solution matches the pH at the point of zero charge (PZC), the surface charge of the photocatalyst becomes neutral, leading to a minimal electrostatic attraction between the photocatalyst surface and ions 80 .

Effect of the photocatalyst dose
This study examines the impact of ZnF-CA-LP NCs concentration on the efficiency of MB degradation following exposure to UV radiation.The dosage of the catalyst varied between 5.0, 10.0 and 20.0 mg.The results of this study indicate that there was an increase in the removal rate when the dose of photocatalyst was increased at 120 min with a range of 71.2% to 93.8% Fig. 9c.It is believed that a higher concentration of photocatalyst in the reaction results in a larger number of active sites on the photocatalyst compared to the volume of the MB solution 81 .

Effect of dye concentration
Figure 9d shows the effect of different concentrations (5.0, 10.0 and 15.0 ppm) of MB dye on the dye degradation as indicated by decolorization, with fixation of other parameters in the reaction.The results demonstrate that decolorization was more effective at a lower dye concentration (5.0 ppm), albeit a substantial decline in decolorization occurring at a greater concentration (15.0 ppm) of dye.This matches with the results of other researchers 82,83 .The suggested explanation is that when the concentration of the dye increases, with a constant concentration of hydroxyl radicals; a lower rate of elimination takes place 84 .The dye molecules must undergo adsorption onto the catalyst prior to interacting with the active iron ion sites on the catalyst.An elevation in dye concentration led to a competition for active sites, resulting in a minor reduction in the percentage of color removal 85 .Furthermore, a high concentration of dye also diminishes the ability of light to penetrate the dye solution, resulting in a decrease in the generation of OH radicals 86 .

Effect of H 2 O 2
In order to investigate the impact of hydrogen peroxide concentrations on the rate of decolorization of MB dye through UV photolysis, a range of H 2 O 2 concentrations were tested to determine its enhancing effect 87 .The process of generating • OH can be accelerated by augmenting the quantity of H 2 O 2 in the initial mixture, as H 2 O 2 serves as a • OH source upon exposure to UV radiation 88 .The present study conducted a thorough investigation on the efficacy of UV/H 2 O 2 in the removal of MB dye.Various concentrations of H 2 O 2 (5, 10 and 15 ppm) were employed, along with a 10 mg sample of ZnF-CA-LP NCs, and a consistent concentration of MB dye (10 ppm at pH 9).The duration of the treatment was set at 10 min.The objective was to identify the optimal circumstances for effectively treating varied quantities of MB dye.The augmentation of H 2 O 2 quantities significantly facilitated the removal of the MB dye.The H 2 O 2 concentrations at 5, 10, and 15 ppm were associated with a corresponding increase in the % removal of MB dye (62.47%, 69.7%, and 79.07%respectively.The removal efficiency of the MB dye increased proportionally with the increase of H 2 O 2 concentration with the increasing of time as depicted in Fig. 10.

Mechanism of photocatalysis of MB
As mentioned in several studies of works of literature [89][90][91] , the possible mechanism is as follows; When photons, in the form of light rays, interact with a material and have energy that is equal to or greater than its bandgap, electrons in the conduction band (CB) move to the valence band (VB) by crossing the bandgap, resulting in the radicals.The possible photocatalytic mechanism is described in Fig. 11.

Reusability of the ZnF-CA-LP NPs
For practical applicability, the reusability of immobilized enzyme is considered an essential component that plays a significant role in making a method economically viable and possible 7 .This suggests that suitable reusability of ZnF-CA-LP NCs in successive cycles of application is an important aspect to study.Therefore, a supplementary investigation was undertaken to examine the potential reusability of ZnF-CA-LP NCs in the context of photocatalytic reduction of MB dye when subjected to UV irradiation.The photocatalytic reuse experiments were conducted using the identical settings as those stated in the earlier evaluation of photocatalytic activity.The dye degradation rates in the initial and subsequent 5 cycles were found to be 85.3%, 77.1%, 65.7%, 59.2%, 51.2%, and 38.3%, respectively as shown in Fig. 12.Before its utilization in the subsequent cycle, ZnF-CA-LP NCs were subjected to centrifugation, followed by washing with deionized water and thereafter allowed to undergo overnight drying.The decrease in residual activity seen over six cycles may be attributed to the agglomeration of nanomaterials, the leaking of enzymes from the supporting materials, and the inactivation of the enzyme 92 .

Kinetic studies
The photocatalytic activity of a catalyst is contingent upon its intrinsic structural characteristics 93 .The degradation kinetics of MB were investigated by utilizing photocatalysts consisting of ZnF-CA-LP NCs under the influence of UV radiation.The rate of MB degradation can be determined using the following Eq.( 5) where (C t and C 0 ) are the respective initial and residual concentrations of MB, t is the removal interval, and k is the removal rate constant.Figure 13.shows the relationship between (− ln C t /C o ) Vs. t (min).The findings showed that pseudo-first-order rate laws were obeyed by the removal process's kinetics.Moreover, the apparent pseudo-first-order rate constants drop with increasing catalyst dosage.

Antibacterial activity
The antibacterial efficacy of the nanoparticles that were synthesized was evaluated by employing the agar well diffusion technique against a single strain of gram-positive bacteria (S. aureus) as well as a single strain of gramnegative bacteria (E.coli).The assessment of ZOI was performed for each bacterial strain about the positive control (GEN), and the negative control, (DMSO).The lack of microbial proliferation in the proximity of NPs can be interpreted as an indirect indication of the NPs' capacity to hinder or suppress microbial growth 94 .The disc agar distribution method, employed as a screening experiment, has revealed that the ZnF-CA-LP nanocomposite   www.nature.com/scientificreports/has a qualitative antibacterial efficacy against the bacteria tested, as depicted in Fig. 14.The results of the in-vitro ZOI demonstrate that ZnF-CA-LP NCs exhibit significant antibacterial activity against S. aureus and E. coli with a ZOI of 16.0 and 20.0 mm, respectively as shown in Table 3.It is observed that the antibacterial efficacy of the ZnF-CA-LP NCs is considerably more than that of ZnF-CA and ZnF NPs, indicating the possible synergistic effect between Lipase and ZnF-CA NPs.The antibacterial capabilities of nanocomposites cannot be only determined by their size; other significant factors such as elemental composition, purity, surface area, and form, require careful consideration 95 .ZnF-CA-LP NCs have several advantages, including a significant surface-to-volume ratio and a nano-scale structure, which facilitate their interaction with biological entities such as bacteria and yeast.The minimum inhibitory concentration (MIC) values of ZnF-CA-LP NCs, ZnF-CA NPs, and ZnF NPs against the different bacteria under study are reported in Table 1 ZnF-CA-LP NCs exhibit significant MIC values of 1.25 μg/mL and 0.625 μg/mL against S. aureus and E. coli, respectively.
Figure 15 shows the antibiofilm activity of ZnF-CA-LP NCs against E. coli bacteria (as a model for susceptible bacteria) using a test tube method.E. coli displayed a concentrated whitish-yellow film that covered the entirety of the interface between air and liquid in the absence of ZnF-CA-LP NCs as shown in Fig. 15a.It demonstrated strong adherence to the inner wall of the test tubes and appeared as a circular blue structure after being stained with crystal Violet (CV), as shown in Fig. 15b.A suspension of blue color was produced by dissolving the ring stained with CV using 99.0%ethanol, Fig. 15c.On the other hand, the test tubes that were inoculated with E.coli and subjected to treatment with ZnF-CA-LP NCs with a concentration of 10.0 µg/ml exhibited a significant inhibitory impact.This was evident from the observed limited proliferation of bacterial rings, as depicted in Fig. 15a.The optical density was measured subsequently at a wavelength of 570.0 nm 99 .Table 3 presents the data on the percentage of inhibition observed in the biofilms generated by the bacteria under investigation.The most significant level of inhibition was seen against E.coli (96.6%), followed by S.aureas (88.4%), during exposure to a concentration of 10.0 µg/ml of the ZnF-CA-LP NCs.The observed variation in the inhibition percentage can be attributed to various factors such as antimicrobial properties, biosorption capacity, physical characteristics, invasive potential, and distinctive chemical properties that regulate the interaction between the nanomaterials and the biofilms 29 .

Mechanism of antimicrobial activity of the synthesized ZnF-CA-LP NCs
Studies have documented the effectiveness of metal nanoparticles as antibacterial agents, and the harmful effects of metals can be evaluated in both bacterial and eukaryotic organisms 100 .Nevertheless, there is a lack of reports   www.nature.com/scientificreports/ on enzyme-metal nanocomposites, and the specific chemical mechanism for their antibacterial action remains unclear 101 .The antibacterial mechanism of metal nanoparticles involves the disruption of the cell membrane, the generation of reactive oxygen species (ROS) that degrade lipids, proteins, or DNA, and the inhibition of bacterial metabolism, ultimately leading to bacterial death.The enzyme-metal nanocomposite exhibits a synergistic effect in dismantling cellular structures and impeding the proliferation of bacteria 102 .
A comprehensive overview was provided for the various processes by which enzyme-metal nanocomposites exert their antibacterial effects, encompassing both extracellular and intracellular pathways, according to the antibacterial properties of nanomaterials.Enzyme-metal nanocomposites, composed of specific hydrolase, can break down extracellular polymeric substrates (EPS) that consist of protein, polysaccharides, and DNA in the external environment.Simultaneously, enzyme-metal nanomaterials can hydrolyze and oxidize peptidoglycan, lipids, and proteins found on the majority of bacterial cell walls and membranes.Enzyme-metal nanocomposites can also produce ROS and disrupt the function of lipids, proteins, and DNA within bacterial cells, hence impacting bacterial metabolism, Fig. 16.

Conclusion
ZnFe 2 O 4 NPs have been synthesized by the green synthesis method using Psidium guava leaf extract as a reducing agent and characterized by structural and optical tools.The surface of ZnFe 2 O 4 NPs was coated with citric acid then the lipase enzyme was immobilized to obtain modified ZnF-CA-LP NCs.The photocatalytic efficiency of the synthesized ZnF-CA-LP NCs was tested against Methylene blue (MB).Also, different parameters affecting the efficiency of removal potential such as pH, MB concentration, and photocatalyst dose have been studied.According to XRD, SEM, and TEM analyses, it was found that ZnF NPs were located at the core, while the citric acid and lipase enzyme were coated on this core, producing ZnF-CA-LP NCs with particle sizes varying from 30.5 nm to 55.5 nm with average 38.50 nm which was larger than the synthesized nacked ZnF NPs due to the loading of CA and lipase on their surface.The ZnF-CA-LP NCs were applied for MB removal as an indicator of its applicability in wastewater treatment.The ZnF-CA-LP NCs had the highest efficiency for MB removal, achieving 96.0% removal of 5.0 ppm of MB at pH 9, and had a reusability for six successive cycles.In addition, we investigated the anti-bacterial activity against S. aureus and E. coli, and demonstrated that ZnF-CA-LP NCs exhibited significant MIC values of 0.312 μg/mL and 0.625 μg/mL against S. aureus and E. coli, respectively.Furthermore, the nanocomposites had a significant antibiofilm activity against S. aureus (88.4%) and E. coli (96.6%).Hence, ZnF-CA-LP NCs are considered promising biocatalysts for potential applications in environmental and biomedical uses.various types of green synthesized metal oxide NPs (ZnO, CuO, TiO 2 , and ZnO/CuO) for different applications need to have more attention in the future.
Figure 4  shows the uniform distribution and characteristic morphology of the synthesized magnetic ZnF NPs, ZnF-CA NPs & ZnF-CA-LP NCs.The synthesis of ZnF NPs was confirmed by analyzing energy-dispersive X-ray spectroscopy EDX test data, which indicated the presence of Fe, Zn, O.The EDX analysis of ZnF-CA NPs revealed the presence of characteristic EDX peaks corresponding to the atoms of Fe, O, Zn, and C which confirm the coating of citric acid.While the EDX spectra and particular SEM of ZnF-CA-LP NCs reveal the presence of characteristic EDX peaks corresponding to the atoms of Fe, O, Zn, C, and N as a confirmation of amide group formation as documented in previous studies67,71,72 .

Figure 7 .
Figure 7. (a) DPPH scavenging activity of ZnF-CA-LP NCs, and (b) UV-Vis spectrum of DPPH at different concentrations with time intervals.

Figure 8 .
Figure 8.(a) UV-Vis spectrum of MB at different concentrations, (b) Calibration curve of different concentrations of MB, (c) The removal due to adsorption in the absence of light, and (d) Removal % of MB under UV photocatalysis using (10 mg nanocatalyst, 50 ml MB solution (10 ppm), Temp.= 25 °C and pH 7.0).

Figure 9 .
Figure 9. (a) Effect of different initial pH values, (b) Point of Zero charge (PZC), (c) Effect of the photocatalyst dose on the removal efficiency of MB (50 ml MB solution (10 ppm), temp.= 25 °C and pH 7.0), and (d) Effect of different MB concentration (5, 10 and 15 ppm) at pH 9.0 and 10.0 mg ZnF-CA-LP NCs.

Figure 10 .
Figure 10.Effect of different concentrations of H 2 O 2 and different time points.

Figure 11 .
Figure 11.Proposed mechanism for the photocatalytic degradation of MB by the synthesized ZnF-CA-LP NCs in the presence of H 2 O 2 .

Figure 15 .
Figure 15.The antibiofilm efficacy of ZnF-CA-LP NCs was evaluated against E. coli utilizing the test tube method.The reported sequence of actions is as follows.(a) The proliferation of bacterial cells and the development of biofilm (rings) in the absence of ZnF-CA-LP NCs treatment, and the suppression of bacterial growth following treatment with ZnF-CA-LP NCs.(b) Application of crystal violet to the bacterial cells that are attached to the surface.(c) Ethanol is used to eliminate and dissolve the attached bacterial and yeast cells to determine the semi-quantitative suppression of biofilm formation (%).

Figure 16 .
Figure 16.The diagram illustrates the five primary mechanisms by which ZnF-CA-LP NCs exhibit their antibacterial properties.(I) The ZnF-CA-LP NCs stick to and attach the surface of the microbial cell, leading to the release of Lipase, which in turn causes damage to the cell membrane and changes in its transport activity.(II) ZnF-CA-LP NCs infiltrate the microbial cells and engage with intracellular biomolecules, thereby influencing the corresponding cellular machinery.(III) The presence of ZnF-CA-LP NCs induces the generation and amplification of ROS, resulting in cellular damage.(IV) The ZnF-CA-LP NCs manipulate the cellular signal pathway and induce cell death.(V) ZnF-CA-LP NCs effectively inhibit the movement of ions into and out of microbial cells.